Frozen gel slicing method and apparatus

ABSTRACT

A method and apparatus for obtaining highly uniform slices of gel containing bands of RNA by freezing the gel in a cylindrical tube and then introducing the frozen cylindrical gel into a cylindrical sample handling mechanism and a gel slicing apparatus. The gel slicing apparatus has a sub-zero brine bath that surrounds the sample handling mechanism to maintain the frozen gel in a frozen state. The gel slicing apparatus includes a crank, drive screw and plunger positioned to drive the frozen sample. The crank is turned one revolution which forces part of the frozen sample out of the sample handling mechanism a predetermined amount. The extended sample is sliced off and then subjected to analysis. The process is then repeated until all of the frozen sample has been sliced.

United States Patent [191 Schaffer et al.

[ FROZEN GEL SLICING It [ETHOD AND APPARATUS [75] Inventors: Frederick L. Schaffer, Moraga;

Marjorie E. Soergel, Berkeley; David C. Straube, San Pablo, all of Calif.

[73] Assignee: The United States of America as represented by the Secretary of the Navy, Washington, DC.

[22] Filed: Feb. 24, 1972 [21] Appl. No.: 229,025

[52] U.S. Cl. 222/146 C, 33/1 P, 83/15, 83/437, 83/9l5.5, 214/310, 222/386, 269/287 [51] Int. Cl. B67d 5/22 [58] Field of Search 83/170, 401, 437, 915.5, 83/15, 698; 269/287; 33/1 F; 22/146 C, 386;

[56] References Cited UNITED STATES PATENTS 2,495,077 1/1950 Protzeller 83/170 K 2,101,755 12/1937 Rosenstone et al 83/437 X FOREIGN PATENTS OR APPLICATIONS 1,245,41 l 8/1971 Great Britain 83/9l5.5

[451 Apr. 30, 1974 883,898 5/1943 France ..83/915.5

Primary Examiner-Andrew R. Juhasz Assistant Examiner\ /V. D. Bray Attorney, Agent, or Firm-R. S. Sciascia; Charles D. B. Curry 57 ABSTRACT A method and apparatus for'obtaining highly uniform slices of gel containing bands of RNA by freezing the gel in a cylindrical tube and then introducing the frozen cylindrical gel into a cylindrical sample handling mechanism and a gel slicing apparatus. The gel slicing apparatus has a sub-zero brine bath that surrounds the sample handling mechanism to maintain the frozen gel in a frozen state. The gel slicing apparatus includes a crank, drive screw and plunger positioned to drive the frozen sample. The crank is turned one revolution which forces part of the frozen sample out of the sample handling mechanism a predetermined amount. The extended sample is sliced off and then subjected to analysis. The process is then repeated until all of the frozen sample has been sliced.

6 Claims, 9 Drawing Figures FROZEN GEL SLICING METHOD AND APPARATUS BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method and apparatus for obtaining small uniform slices from a sample of frozen gel.

2. Description of the Prior Art Gel electrophoresis is well known as a technique for separating different ribonucleic acids (RNA) from each other. The process involves'placing a column of gel and an unknown sample of RNA on top of it into a bath and subjecting them to an electrophoretic pro cess wherein the RNA forms bands of different RNAs in the gel. The position of each band of RNA in the gel depends upon the migration rate of the RNA in the gel which is inversely proportional to the molecular weight of the different RNAs. By very accurate and uniform slicing of the gel, containing bands of separated RNA, it is possible to determine the molecular weight of the RNA in that particular slice or band by comparing it with known RNAs. Gels of low concentration, used to separate RNAs of high molecular weight, lack rigidity and are difficult to handle. One technique used for providing some increased rigidity of the gel has been the incorporation of agarose as a stiffener. This has the disadvantage of increased complexity of preparation. An alternative technique has been the use of a dialysis membrane to retain the soft gel in vertical tube electrophoresis. Most procedures require removal of the soft gel from the tube for sectioning and analysis. Because of the mechanical properties of the gel there are difficulties in removal and sectioning in the unfrozen state. A typical procedure has been to remove the gel from the tube and then freeze and slice the frozen gel. This has the disadvantage of introducing a non-uniform sagging of the soft gel after it has been removed from the tube and before it has been frozen. In addition, an unrestrained sample of frozen gel is difficult to properly handle for slicing and may require the use of a plurality of parallel knife blades to simultaneously slice the sample. This involves the use of expensive apparatus which is difficult to operate, is easily damaged, and still results in non-uniform samples especially due to the nonuniform sagging.

The present invention overcomes these disadvantages by freezing the gel prior to removal from the tube and introducing the frozen gel into a slicing apparatus which retains a high degree of uniformity of the sliced samples.

SUMMARY OF THE INVENTION Briefly, the present invention comprises using a dialysis membrane to retain the soft gel in vertical tube electrophoresis, freezing the gel while still in the tube, and inserting the frozen gel sample into the gel slicing apparatus of the present invention. The frozen gel slicing apparatus comprises a base having a crank and drive screw' mounted at one end and a cylindrical sample handling mechanism and brine bath container at the other. The sample handling mechanism is mounted within the brine bath container and a plunger interconnects the drive screw and the sampling handling mechanism which are in axial alignment. The frozen cylindrical sample is inserted into the sample handling container and is maintained frozen by sub-zero brine. The

crank is turned one revolution which forces part of the sample out from thesample handling mechanism a predetermined amount. The extended sample is sliced off and then subjected to analysis. The process is then repeated until all of the frozen sample has been sliced.

STATEMENT OF THE OBJECTS OF INVENTION conjunction with the accompanying drawings wherein:

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a pictorial view, partly in section, of the frozen gel slicing apparatus of the present'invention;

FIG. 2 is a side elevation view of the sample handling mechanism of FIG. 1;

FIG. 3-is a schematic drawing illustrating a conven-f tional method of vertical tube electrophoresis;

FIG. 4 is side elevation of the sample contained in the liquid gel from the method of FIG. 3;

FIG. 5 is a side elevation of the freezer and the frozen gel sample of FIG. 4; and V FIGS. 6A, 6B, 6C, and 6D are schematic drawings illustrating the process steps of the apparatus of the present invention. 1

DESCRIPTION OF THE PREFERRED EMBODIMENTS In FIG. 1 is a pictorial view, partly in section, of the frozengel slicing apparatus 11 of the present invention. The apparatus includes a base 13 that is supported by legs 15 and 17. At one end of the base is a crank holding section 19 including a lower support 21, an upper support 23, holding screws and a female threaded member, not shown. The female threaded member may be 'a square nut, for example that is held between lower support 21 and uppersupport 23 by holding screws 25. An elongated drive screw 29 is in threaded engagement with the female threaded member and is rotated by means of hand crank 31 which is attached to one end thereof. The other end of drive screw 29 includes a rubber or plastic sleeve 33 which receives one end of plunger 35 which is free to rotate within sleeve 33 and abuts the end of drive screw 29.

In FIGS. 1 and 2 is illustrated sample handling mechariisin 37 which includes a precision bore tube 39, two conical rubber or plastic sleeves 41 and 43 and sleeve 45. Sleeves 41 and 43 seal sample handling mechanism 37 into brine bath container 47. Plunger 35 has a rubber end piece 49 connected to the end thereof. End piece 49 is generally cylindrical in shape having a close fitting end 51. Sleeve 45 is inserted on the end of tube 39 and is frictionally held in placeby clip shaped sup-' port member 53. Referring to FIG. 1, container 47 is held in place by corner supports and 57 and by inserting sleeve 45 into clip shaped support member 53. Plunger 35 may be disengaged from drive screw 29 and rubber sleeve 33 by sliding it forward. Plunger 35 may be then lifted upwards and withdrawn from precision 3 bore tube 39. Container 47,'including sample handling mechanism 37,.ma'y be then removed from base 13 by lifting and thereby disengaging sleeve 45 from support member 53.

In FIG. 3 is illustrated a conventional method of vertical tube electrophoresis. A soft gel 598 is retained in precisionbore holding'tube 61 by a dialysis membrane 62. The membrane is retained in place by means of a plastic sleeve 65. The soft gel 59S was obtained from a liquid gel material as a result of a previously performed conventional polymerization process. A separator 67 is attached to the walls of a dielectric container 69 to separate buffer solution 71 from buffer solution 73. A sample 75 of a plurality of unknown RNAs is placed on the top of soft gel 598. Conical rubber sleeve 77 surrounds tube 61 to provide a seal. Power is then supplied to negative electrode 79 and positive electrode 81 by a direct current power source, such as battery 83. The sample 75 of RNAs then migrate downward and into the soft gel 57. The migration continues until the sample is distributed as a plurality of disc shaped bands in the soft gel. The position of each band of RNA in the gel'depends upon the migration rate of the RNA in the gel which is inversely proportionalto the molecular weight of the different RNAs. By very accurate and uniform slicing of the gel, containing bands of separated RNAs, it is possible to determine the molecular weight of the RNA in that particular slice by comparing it with known RNA. After the RNA migration is completed, the power is turned off and the tube 61 is removed from container 69 and sleeve 77 is ,alsofremove'd. As illustrated in-FIG. 4, and in accordance with the present invention, a glass push rod 85,

having a flat lower end that has a diameterslightly less than that ofv tube 61, is inserted into tube 61 and into facial contact with the upper surface of soft gel 59S. The flat lower end of glass push rod 85 forms the upper end of soft gel 595 into a flat surface to optimize the accuracy of the initial gel slices as hereinafter described.

' is then stored in freezer 87 of FIG. at about 20C.

In FIGS. 6A, 6B, 6C, and 6D are schematic drawings illustrating the operating sequence of the process and apparatus of the present invention using the frozen gel 59F of FIG. 5.

In FIG. 6A is shown the apparatus ready to receive the frozen sample 59F that is contained in freezer 87 of FIG. 5. For initial loading the container 47 is cooled to about 20C.

In FIG. 6B is illustrated holding tube 61, containing frozen sample 59F, operatively connected to tube 39 by means of collar 65 and in precise alignment therewith. Plunger 49 is positioned flush with the end of tube 39 to eliminate the formation of an air pocket between gel 59F and plunger 49. Holding tube 61 and tube 39 are both precision bore tubes of the same diameter which is essential for successful operation. Push rod 85 is flat against frozen sample 59F and IS pushed to the left, as indicated by the arrow, by means of another push rod 89. Prior to loading the frozen holding tube 4 61 it is briefly'immersed in water to loosen the still frozen gel from the tube.

As illustrated in FIG. 6C, after sample 59F is pushed into tube 39 the empty holding tube 6l is removed and a'sub zero brine solution 63, at about 5C, is added. From this itcan be seen that frozen sample 59F is now contained in tube 39 and is prevented from thawing out by the surrounding sub-zero brine.

As shown in FIG. 6D, plunger 35 is moved to the right by turning hand crank 31 of FIG. 1. The plunger is moved to the right forcing the sample to the right, as shown by the arrow in FIG. 6D. The frozen gel is moved to the right until the gel first extends beyond the tube 39. The sample is then sliced off, with knife 58 of FIG. 1, for example, along dotted line 91 of FIG. 6D.

The hand crank 31 is then turned one full turn and the sample is again cut. Each slice'is put into a container for subsequent analysis. This process is repeated until all of frozen sample 59F has been sliced. A drive screw having 16 threads per inch provides a sample slice of about 1.6 millimeters which is satisfactory for most purposes.

We claim: 1

1. An apparatus for slicing frozen gel comprising:

a. a container;

b. a sample handlingmechanism including an open tube having first and second ends and first and second sealing means;

c. said sample handling mechanism being mounted in said container wherein said first end extends through the wall of said container andsaid second end extends through another part of the wall of said container;

d. points intermediate of said first and second ends of said tube are respectively sealed to said container wall by said first and second sealingmeans;

e. an elongated plunger for being inserted into and withdrawn from said one end of said tube;

f. means for moving said plunger into said tube by sequential and uniformincrem'ents and I g. said container contains brine at a sub-zero temperature.

2. The apparatus of claim 1 including:

a. afrozen samplepositioned in said tube of said handling mechanism and being kept in a frozen state by said brine.

3. The apparatus of claim 2 wherein:

a. said second end of said tube is flat; and b. said means moves said plunger and said plunger moves said sample out through said second end of i d. the other end of said plunger being removably connected to one end of said drive screw.

6. The apparatus of claim 5 including: i

a. a support membenmounted on said base;

b. said support member comprising a-pair of elongated flared out pins for receiving one end of said tube of said sample handling mechanism.

* it l l 

1. An apparatus for slicing frozen gel comprising: a. a container; b. a sample handling mechanism including an open tube having first and second ends and first and second sealing means; c. said sample handling mechanism being mounted in said container wherein said first end extends through the wall of said container and said second end extends through another part of the wall of said container; d. points intermediate of said first and second ends of said tube are respectively sealed to said container wall by said first and second sealing means; e. an elongated plunger for being inserted into and withdrawn from said one end of said tube; f. means for moving said plunger into said tube by sequential and uniform increments and g. said container contains brine at a sub-zero temperature.
 2. The apparatus of claim 1 including: a. a frozen sample positioned in said tube of said handling mechanism and being kept in a frozen state by said brine.
 3. The apparatus of claim 2 wherein: a. said second end of said tube is flat; and b. said means moves said plunger and said plunger moves said sample out through said second end of said tube by sequential and uniform amounts.
 4. The apparatus of claim 1 wherein: a. one end of said plunger has connected thereto a rubber end piece.
 5. The apparatus of claim 4 including: a. a base; b. said container removably mounted at one end of said base; c. said means includes an elongated drive screw rotatably mounted at the other end of said base; and d. the other end of said plunger being removably connected to one end of said drive screw.
 6. The apparatus of claim 5 including: a. a support member mounted on said base; b. said support member comprising a pair of elongated flared out pins for receiving one end of said tube of said sample handling mechanism. 